Methods and Compositions for Detection of Ehrlichia chaffeensis (p120)

ABSTRACT

The invention provides methods and compositions for the detection of  Ehrlichia chaffeensis.

PRIORITY

This application claims the benefit of U.S. Ser. No. 60/974,203, filedon Sep. 21, 2007, and U.S. Ser. No. 60/974,601, filed on Sep. 24, 2007,both of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The Ehrlichia are obligate intracellular pathogens that infectcirculating lymphocytes in mammalian hosts. Ehrlichia canis andEhrlichia chaffeensis are members of the same sub-genus group thatinfect canines and humans and can each cause canine monocyticehrlichiosis (CME) and human monocytic ehrlichiosis (HME), respectively.The canine disease is characterized by fever, lymphadenopathy, weightloss, and pancytopenia. In humans the disease is characterized by fever,headache, mylagia, and leukopenia. Early detection and treatment areimportant for treating both canine and human ehrlichiosis.

Indirect immunofluorescense assays (IFA) and enzyme-linked immunosorbentassays (ELISA) are frequently used as aids in the diagnosis of thesediseases. These assays measure or otherwise detect the binding ofanti-Ehrlichia antibodies from a patient's blood, plasma, or serum toinfected cells, cell lysates, or purified Ehrlichia proteins. However,many assays for detecting anti-Ehrlichia chaffeensis antibodies orfragments thereof are severely limited in usefulness because ofsensitivity and specificity issues directly related to the impure natureof the Ehrlichia antigen used in these tests. Additionally, animalsvaccinated for E. canis may show a positive result when tested for E.chaffeensis due to immunological cross-reaction. Highly purified,specific reagents are needed to construct more accurate assays.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a purified polypeptidecomprising SEQ ID NO:1, wherein the polypeptide consists of less thanabout 50 contiguous naturally occurring Ehrlichia chaffeensis aminoacids; SEQ ID NO:2, wherein the polypeptide consists of less than about50 contiguous naturally occurring Ehrlichia chaffeensis amino acids; SEQID NO:4, wherein the polypeptide consists of less than about 50contiguous naturally occurring Ehrlichia chaffeensis amino acids; SEQ IDNO:5, wherein the polypeptide consists of less than about 50 contiguousnaturally occurring Ehrlichia chaffeensis amino acids. The purifiedpolypeptide can consist of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:5. The invention also provides an isolated polynucleotide thatencodes the purified polypeptides of the invention. A purifiedpolypeptide of the invention can be linked to an indicator reagent, anamino acid spacer, an amino acid linker, a signal sequence, a stoptransfer sequence, a transmembrane domain, a protein purificationligand, a heterologous polypeptide, one or more additional polypeptidescomprising SEQ ID NOs:1, 2, 3, 4, 5, or a combination thereof. Thepurified polypeptide can comprise one or more C amino acid residues atthe amino terminus or carboxy terminus or both termini of thepolypeptide.

Another embodiment of the invention provides a method of detectingantibodies that specifically bind an Ehrlichia chaffeensis polypeptidein a test sample. The method comprises contacting a purified polypeptidecomprising SEQ ID NO:1, 2, 3, 4, or 5 with the test sample, underconditions that allow polypeptide/antibody complexes to form. When thepurified polypeptide comprises SEQ ID NO:1, 2, 4 or 5, the purifiedpolypeptide consists of less than about 50 contiguous naturallyoccurring Ehrlichia chaffeensis amino acids. When the purifiedpolypeptide comprises SEQ ID NO:3, the purified polypeptide consists ofless than about 575 contiguous naturally occurring Ehrlichia chaffeensisamino acids. The polypeptide/antibody complexes are detected. Thedetection of the polypeptide/antibody complexes is an indication thatantibodies specific for Ehrlichia chaffeensis are present in the testsample, and the absence of the polypeptide/antibody complexes is anindication that antibodies specific for Ehrlichia chaffeensis are notpresent in the test sample. The complexes can be contacted with anindicator reagent prior to the detection step. In one embodiment of theinvention, the purified polypeptide is SEQ ID NOs:1 2, 4, or 5 and themethod does not detect antibodies that specifically bind an Ehrlichiacanis polypeptide. The amount of antibody in the test sample can bedetermined. The purified polypeptide can be attached to a substrate. Thepurified polypeptide can be linked to an indicator reagent, an aminoacid spacer, an amino acid linker, a signal sequence, a stop transfersequence, a transmembrane domain, a protein purification ligand, aheterologous protein, one or more additional polypeptides comprising SEQID NOs:1, 2, 3, 4, 5 or a combination thereof.

Yet another embodiment of the invention provides a method of detectingan Ehrlichia chaffeensis infection in a subject. The method comprisesobtaining a biological sample from the subject; contacting a purifiedpolypeptide comprising SEQ ID NO:1, 2, 3, 4 or 5 with the biologicalsample under conditions that allow polypeptide/antibody complexes toform. When the purified polypeptide comprises SEQ ID NO:1, 2, 4 or 5,the purified polypeptide consists of less than about 50 contiguousnaturally occurring Ehrlichia chaffeensis amino acids. When the purifiedpolypeptide comprises SEQ ID NO:3, the purified polypeptide consists ofless than about 575 contiguous naturally occurring Ehrlichia chaffeensisamino acids. The polypeptide/antibody complexes are detected. Thedetection of the polypeptide/antibody complexes is an indication thatthe subject has an Ehrlichia chaffeensis infection and the absence ofthe polypeptide/antibody complexes is an indication that the subjectdoes not have an Ehrlichia chaffeensis infection. In one embodiment ofthe invention, the purified polypeptide is SEQ ID NO:1, 2, 4, or 5 andthe method does not detect Ehrlichia canis infection in the subject.

Another embodiment of the invention provides an antibody thatspecifically binds to a polypeptide consisting of SEQ ID NO:1, 2, 4 or5. The antibody can be a monoclonal antibody, polyclonal antibody,antigen-binding antibody fragment, or a single chain antibody.

Still another embodiment of the invention provides a method of detectingan Ehrlichia chaffeensis polypeptide in a sample. The method comprisescontacting antibodies that specifically bind to a polypeptide consistingof SEQ ID NO:1 2, 4, or 5 with the sample under conditions that allowpolypeptide/antibody complexes to form; and detecting thepolypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that an Ehrlichiachaffeensis polypeptide is present in the sample and the absence of thepolypeptide/antibody complexes is an indication that an Ehrlichiachaffeensis polypeptide is not present in the sample. The antibodies canbe monoclonal antibodies, polyclonal antibodies, antigen-bindingantibody fragments, or single chain antibodies. The antibodies can beattached to a substrate.

Therefore, the invention provides methods and compositions to detect E.chaffeensis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of serum assays of dogs that wereexperimentally infected with E. chaffeensis. p120B (SEQ ID NO:4) (shownas “IDX P120” in the figures) and p120-R (SEQ ID NO:3) (shown as“P120-R” in the figures) both were able to detect E. chaffeensisantibodies by day 7 post-infection. For canine CTUALJ on day 7 thesample was PCR positive for E. chaffeensis and the IFA titer was 1:80.On day 96, which was post-booster, the IFA titer was 1:5120. For canineCURALN on day 7 the sample was PCR positive for E. chaffeensis and theIFA titer was 1: 160. On day 82, which was post-booster, the IFA titerwas 1:1280.

DETAILED DESCRIPTION OF THE INVENTION

Ehrlichia chaffeensis Polypeptides

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

A polypeptide is a polymer of two or more amino acids covalently linkedby amide bonds. A polypeptide can be post-translationally modified. Apurified polypeptide is a polypeptide preparation that is substantiallyfree of cellular material, other types of polypeptides, chemicalprecursors, chemicals used in synthesis of the polypeptide, orcombinations thereof. A polypeptide preparation that is substantiallyfree of cellular material, culture medium, chemical precursors,chemicals used in synthesis of the polypeptide, etc., has less thanabout 30%, 20%, 10%, 5%, 1% or more of other polypeptides, culturemedium, chemical precursors, and/or other chemicals used in synthesis.Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% ormore pure. A purified polypeptide does not include unpurified orsemi-purified cell extracts or mixtures of polypeptides that are lessthan 70% pure.

The term “polypeptides” can refer to one or more of one type ofpolypeptide (a set of polypeptides). “Polypeptides” can also refer tomixtures of two or more different types of polypeptides (a mixture ofpolypeptides). The terms “polypeptides” or “polypeptide” can each alsomean “one or more polypeptides.”

One embodiment of the invention provides a purified Ehrlichiachaffeensis polypeptide as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:5. An X stands for any amino acid.

TEREXEIESHQGETEKESGITESHQKEDEIVSQX SEQ ID NO:1

In one embodiment the X at position 5 of SEQ ID NO:1 is S or N and the Xat position 34 is P or S.

KESGITESHQKEDEIVSQX SEQ ID NO:2

In one embodiment the X at position 19 of SEQ ID NO:2 is S or P.

MDIDNSNIST ADIRSNTDGL IDIIMRILGF GNKNIVQPQD LGSEIYQQEQ EDDTVSQPSLEPFVAESEVS KVEQEKTNPE VLIKDLQDVA SHESGVSDQP AQVVTERENE IESHQGETEKESGITESHQK EDEIVSQSSS EPFVAESEVS KVEQEETNPE VLIKDLQDVA SHESGVSDQPAQVVTEREXE IESHQGETEK ESGITESHQK EDEIVSQXSS EPFVAESEVS KVEQEETNPEVLIKDLQDVA SHESGVSDQP AQVVTERESE IESHQGETEK ESGITESHQK EDEIVSQPSSEPFVAESEVS KVEQEETNPE VLIKDLQDVA SHESGVSDQP AQVVTERESE IESHQGETEKESGITESHQK EDEIVSQPSS EPFVAESEVS KVEQEKTNPE ILVEDLPLGQ VIPVVVEKDEMFAPSFNPIV IKEEDKVCET CEQEFEIVKD SQTVKGSEDI ISPMQCLESM DSIVSTIFESGMLCPMSKPG QYVCGYEMYM YGFQDVKDLL GGLLSNVPVC CNVSLYFMEH NYFTNHENINHNVVNDIV SEQ ID NO:3  (p120-R)In one embodiment the X at position 189 is an S or N and the X atposition 218 is an S or P.

Each of SEQ ID NOs:1-3 may have an N-terminal C residue. Alternatively,the N-terminal C residue can be absent. Polypeptide P120B is SEQ ID NO:1with an amino terminal C residue where the X at position 5 of SEQ IDNO:1 is S and the X at position 34 of SEQ ID NO:1 is P (i.e.,CTERESEIESHQGETEKESGITESHQKEDEIVSQP; SEQ ID NO:4). Polypeptide p120BK isSEQ ID NO:2 with an amino terminal C residue where the X at position 19of SEQ ID NO:2 is P (i.e., CKESGITESHQKEDEIVSQP; SEQ ID NO:5).

One embodiment of the invention provides a purified polypeptidecomprising SEQ ID NO:1-5, wherein the polypeptide consists of less thanabout 650, 625, 600, 575, 550, 548, 525, 500, 450, 400, 350, 300, 250,200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10or less (or any range between 650 and 10) contiguous naturally occurringEhrlichia chaffeensis amino acids. In one embodiment of the invention apurified polypeptide comprises SEQ ID NO:1-5, wherein the polypeptidecomprises more than about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 525, 548,550, 575, 600, 625, or 650 contiguous naturally occurring Ehrlichiachaffeensis amino acids (or any range between about 10 and 650 aminoacids). In one embodiment of the invention a purified polypeptideconsists of less than about 30, 40, 50, 60, 70, 80, 90, or 100, 125,150, 175, 200, 250, 300, 350, 400, 450, 500, 525, 548, 550, 575, 600,625, 650 contiguous naturally occurring Ehrlichia chaffeensis aminoacids (or any range between 30 and 650) (i.e., the purified polypeptidedoes not encompass the entire naturally occurring Ehrlichia chaffeensisp120 polypeptide. Naturally occurring Ehrlichia chaffeensis amino acidsare any polypeptides naturally produced by an Ehrlichia chaffeensisorganism. That is, a purified polypeptide comprises a polypeptide shownin SEQ ID NOs:1-5, but consists of less than about 650, 625, 600, 575,550, 548, 525, 500, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100,90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 contiguous naturally occurringEhrlichia chaffeensis amino acids (or any range between 650 and 20 aminoacids).

The fact that polypeptides SEQ ID NOs:1-5 are smaller than the fulllength Ehrlichia chaffeensis polypeptide p120 is important becausesmaller polypeptides can have greater specificity and/or sensitivitythan full length polypeptides assays. Additionally, these smallerpolypeptides can be less expensive to manufacture, and may be obtainedat greater purity than the full length polypeptide.

One embodiment of the invention provides a purified polypeptide that isless than about 548, 525, 500, 450, 400, 350, 300, 250, 200, 175, 150,125, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 contiguous naturallyEhrlichia chaffeensis amino acids and greater than about 10, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400,450, 500, 525, or 548 contiguous amino acids of SEQ ID NOs:1-5 (or anyrange between 548 and 20 amino acids).

One embodiment of the invention provides a purified polypeptidecomprising at least about 10, 20, 25, 30, 35, 40, 50 or more contiguousamino acids of SEQ ID NOs:1-5. Therefore, a polypeptide of the inventioncan be, for example, about 19 to about 40; about 19 to about 50; about19 to about 100; or about 19 to about 150 amino acids in length. In oneembodiment of the invention, the polypeptide comprises from about aminoacid residue 85 to about amino acid residue 160 of SEQ ID NO:3; fromabout amino acid residue 90 to about amino acid residue 150 of SEQ IDNO:3; or from about amino acid residue 100 to about 140 of SEQ ID NO:3.

Variant polypeptides are at least about 79% or 80%, or about 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to the polypeptide sequences shown in SEQ ID NOs:1-5 and arealso polypeptides of the invention. For example, a variant polypeptideof SEQ ID NO:1 can be about at least 97% (about 1 amino acid change),94% (about 2 amino acid changes), 91% (about 3 amino acid changes), 88%(about 4 amino acid changes), 85% (about 5 amino acid changes), 82%(about 6 amino acid changes), or about 79% (about 7 amino acid changes)identical to SEQ ID NO:1. A variant polypeptide of SEQ ID NO:2 can beabout at least 95% (about 1 amino acid change), 89% (about 2 amino acidchanges), 84% (about 3 amino acid changes), or 79% (about 4 amino acidchanges) identical to SEQ ID NO:2. A variant polypeptide of SEQ ID NO:4can be about at least 97% (about 1 amino acid change), 94% (about 2amino acid changes), 91% (about 3 amino acid changes), 89% (about 4amino acid changes), 85% (about 5 amino acid changes), 83% (about 6amino acid changes), or about 80% (about 7 amino acid changes) identicalto SEQ ID NO:4. A variant polypeptide of SEQ ID NO:5 can be about atleast 95% (about 1 amino acid change), 90% (about 2 amino acid changes),85% (about 3 amino acid changes), or 80% (about 4 amino acid changes)identical to SEQ ID NO:5.

Variant polypeptides have one or more conservative amino acid variationsor other minor modifications and retain biological activity, i.e., arebiologically functional equivalents. A biologically active equivalenthas substantially equivalent function when compared to the correspondingwild-type polypeptide. In one embodiment of the invention a polypeptidehas about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or less conservative aminoacid substitutions.

Percent sequence identity has an art recognized meaning and there are anumber of methods to measure identity between two polypeptide orpolynucleotide sequences. See, e.g., Lesk, Ed., Computational MolecularBiology, Oxford University Press, New York, (1988); Smith, Ed.,Biocomputing: Informatics And Genome Projects, Academic Press, New York,(1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data,Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence AnalysisIn Molecular Biology, Academic Press, (1987); and Gribskov & Devereux,Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991).Methods for aligning polynucleotides or polypeptides are codified incomputer programs, including the GCG program package (Devereux et al.,Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) whichuses the local homology algorithm of Smith and Waterman (Adv. App.Math., 2:482-489 (1981)). For example, the computer program ALIGN whichemploys the FASTA algorithm can be used, with an affine gap search witha gap open penalty of −12 and a gap extension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Variant polypeptides can generally be identified by modifying one of thepolypeptide sequences of the invention, and evaluating the properties ofthe modified polypeptide to determine if it is a biological equivalent.A variant is a biological equivalent if it reacts substantially the sameas a polypeptide of the invention in an assay such as animmunohistochemical assay, an enzyme-linked immunosorbent Assay (ELISA),a radioimmunoassay (RIA), immunoenzyme assay or a western blot assay,e.g. has 90-110% of the activity of the original polypeptide. In oneembodiment, the assay is a competition assay wherein the biologicallyequivalent polypeptide is capable of reducing binding of the polypeptideof the invention to a corresponding reactive antigen or antibody byabout 80, 95, 99, or 100%. An antibody that specifically binds acorresponding wild-type polypeptide also specifically binds the variantpolypeptide.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his.

A polypeptide of the invention can further comprise a signal (or leader)sequence that co-translationally or post-translationally directstransfer of the protein. The polypeptide can also comprise a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide can beconjugated to an immunoglobulin Fc region or bovine serum albumin.

A polypeptide can be covalently or non-covalently linked to an aminoacid sequence to which the polypeptide is not normally associated within nature, i.e., a heterologous amino acid sequence. A heterologousamino acid sequence can be from a non-Ehrlichia chaffeensis organism, asynthetic sequence, or an Ehrlichia chaffeensis sequence not usuallylocated at the carboxy or amino terminus of a polypeptide of theinvention. Additionally, a polypeptide can be covalently ornon-covalently linked to compounds or molecules other than amino acids,such as indicator reagents. A polypeptide can be covalently ornon-covalently linked to an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, or a combination thereof. A polypeptide canalso be linked to a moiety (i.e., a functional group that can be apolypeptide or other compound) that enhances an immune response (e.g.,cytokines such as IL-2), a moiety that facilitates purification (e.g.,affinity tags such as a six-histidine tag, trpE, glutathione, maltosebinding protein), or a moiety that facilitates polypeptide stability(e.g., polyethylene glycol; amino terminus protecting groups such asacetyl, propyl, succinyl, benzyl, benzyloxycarbonyl ort-butyloxycarbonyl; carboxyl terminus protecting groups such as amide,methylamide, and ethylamide). In one embodiment of the invention aprotein purification ligand can be one or more C amino acid residues at,for example, the amino terminus or carboxy terminus or both termini of apolypeptide of the invention. An amino acid spacer is a sequence ofamino acids that are not associated with a polypeptide of the inventionin nature. An amino acid spacer can comprise about 1, 5, 10, 20, 100, or1,000 amino acids.

If desired, a polypeptide of the invention can be part of a fusionprotein, which can also contain other amino acid sequences, such asamino acid linkers, amino acid spacers, signal sequences, TMR stoptransfer sequences, transmembrane domains, as well as ligands useful inprotein purification, such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A. More than one polypeptide of the inventioncan be present in a fusion protein of the invention. A polypeptide ofthe invention can be operably linked to non-Ehrlichia chaffeensisproteins or non-Ehrlichia chaffeensis p120 proteins to form fusionproteins. A fusion protein of the invention can comprise one or more ofEhrlichia chaffeensis polypeptides of the invention, fragments thereof,or combinations thereof. A fusion protein does not occur in nature. Theterm “operably linked” means that the polypeptide of the invention andthe other polypeptides are fused in-frame to each other either to theN-terminus or C-terminus of the polypeptide of the invention.

Polypeptides of the invention can be in a multimeric form. That is, apolypeptide can comprise one or more copies of an Ehrlichia chaffeensispolypeptide of the invention or a combination thereof. A multimericpolypeptide can be a multiple antigen peptide (MAP). See e.g., Tam, J.Immunol. Methods, 196:17-32 (1996).

Polypeptides of the invention can comprise an antigen that is recognizedby an antibody specific for Ehrlichia chaffeensis. The antigen cancomprise one or more epitopes (i.e., antigenic determinants). An epitopecan be a linear epitope, sequential epitope or a conformational epitope.Epitopes within a polypeptide of the invention can be identified byseveral methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf,CABIOS 4:181-186 (1988). For example, a polypeptide of the invention canbe isolated and screened. A series of short peptides, which togetherspan an entire polypeptide sequence, can be prepared by proteolyticcleavage. By starting with, for example, 30-mer polypeptide fragments(or smaller fragments), each fragment can be tested for the presence ofepitopes recognized in an ELISA. For example, in an ELISA assay anEhrlichia chaffeensis polypeptide, such as a 30-mer polypeptidefragment, is attached to a solid support, such as the wells of a plasticmulti-well plate. A population of antibodies are labeled, added to thesolid support and allowed to bind to the unlabeled antigen, underconditions where non-specific absorption is blocked, and any unboundantibody and other proteins are washed away. Antibody binding isdetected by, for example, a reaction that converts a colorless substrateinto a colored reaction product. Progressively smaller and overlappingfragments can then be tested from an identified 30-mer to map theepitope of interest.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used. Optionally, a polynucleotide encoding a polypeptidecan be translated in a cell-free translation system. A polypeptide canalso be chemically synthesized or obtained from Ehrlichia chaffeensiscells.

An immunogenic polypeptide of the invention can comprise an amino acidsequence shown in SEQ ID NOs:1-5 or fragments thereof. An immunogenicpolypeptide can elicit antibodies or other immune responses (e.g.,T-cell responses of the immune system) that recognize epitopes of apolypeptide having SEQ ID NOs:1-5. An immunogenic polypeptide of theinvention can also be a fragment of a polypeptide that has an amino acidsequence shown in SEQ ID NOs:1-5. An immunogenic polypeptide fragment ofthe invention can be about 6, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 300, 400, 500, or more amino acids in length (or anyrange between 6 and 500). An immunogenic polypeptide fragment of theinvention can be about 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50,40, 30, 20, 15, 10, 6, or less amino acids in length (or any rangebetween 500 and 6).

Ehrlichia chaffeensis Polynucleotides

Polynucleotides of the invention contain less than an entire microbialgenome and can be single- or double-stranded nucleic acids. Apolynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. A nucleic acid moleculeexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest are not to be considered an isolatedpolynucleotide. The polynucleotides of the invention encode thepolypeptides of the invention described above. In one embodiment of theinvention the p120 polynucleotides encode a polypeptide shown in SEQ IDNOs:1-5 or fragments thereof.

Polynucleotides of the invention can consist of less than about 1,500,1,000, 500, 400, 360, 300, 250, 200, 150, 120, 100, 90, 75, 60, 57, or54 (or any range between 1,500 and 54) contiguous, naturally occurringEhrlichia chaffeensis polynucleotides. Polynucleotides of the inventioncan consist of greater than about 54, 57, 60, 75, 90, 100, 120, 150,200, 250, 300, 360, 400, 500, 1,000, 1,500 (or any range between 54 and1,500), or more contiguous, naturally occurring Ehrlichia chaffeensispolynucleotides. The purified polynucleotides can comprise additionalheterologous nucleotides (that is, nucleotides that are not fromEhrlichia chaffeensis) and even additional Ehrlichia chaffeensis aminoacids as long as they do not naturally occur contiguously with Ehrlichiachaffeensis p120 polynucleotides. Polynucleotides of the invention cancomprise other nucleotide sequences, such as sequences coding forlinkers, signal sequences, TMR stop transfer sequences, transmembranedomains, or ligands useful in protein purification such asglutathione-S-transferase, histidine tag, and Staphylococcal protein A.One embodiment of the invention provides a purified polynucleotidecomprising at least about 6, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200,300, 400, 500, 600, 700, 800, 900, 1,000, 1,500 or more contiguousnucleotides of encoding SEQ ID NOs:1-5.

Polynucleotides of the invention can be isolated. An isolatedpolynucleotide is a naturally-occurring polynucleotide that is notimmediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences that it is naturally associated with. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules.

Polynucleotides of the invention can also comprise fragments that encodeimmunogenic polypeptides. Polynucleotides of the invention can encodefull-length polypeptides, polypeptide fragments, and variant or fusionpolypeptides.

Degenerate nucleotide sequences encoding polypeptides of the invention,as well as homologous nucleotide sequences that are at least about 80,or about 90, 96, 98, or 99% identical to the polynucleotide sequences ofthe invention and the complements thereof are also polynucleotides ofthe invention. Percent sequence identity can be calculated as describedin the “Polypeptides” section. Degenerate nucleotide sequences arepolynucleotides that encode a polypeptide of the invention or fragmentsthereof, but differ in nucleic acid sequence from the wild-typepolynucleotide sequence, due to the degeneracy of the genetic code.Complementary DNA (cDNA) molecules, species homologs, and variants ofEhrlichia chaffeensis polynucleotides that encode biologicallyfunctional Ehrlichia chaffeensis polypeptides also are Ehrlichiachaffeensis polynucleotides.

Polynucleotides of the invention can be isolated from nucleic acidsequences present in, for example, a biological sample, such as blood,serum, saliva, or tissue from an infected individual. Polynucleotidescan also be synthesized in the laboratory, for example, using anautomatic synthesizer. An amplification method such as PCR can be usedto amplify polynucleotides from either genomic DNA or cDNA encoding thepolypeptides.

Polynucleotides of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, polynucleotides can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Polynucleotides of the invention can be used, for example, as probes orprimers, for example, PCR primers, to detect the presence of Ehrlichiachaffeensis polynucleotides in a test sample, such as a biologicalsample. Probes are molecules capable of interacting with a targetnucleic acid, typically in a sequence specific manner, for example,through hybridization. Primers are a subset of probes that can supportan enzymatic manipulation and that can hybridize with a target nucleicacid such that the enzymatic manipulation occurs. A primer can be madefrom any combination of nucleotides or nucleotide derivatives or analogsavailable in the art that do not interfere with the enzymaticmanipulation.

A probe or primer can be about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100 or more contiguous nucleotides that encode polypeptides shown inSEQ ID NOs:1-5.

The hybridization of nucleic acids is well understood in the art anddiscussed herein. Typically a probe can be made from any combination ofnucleotides or nucleotide derivatives or analogs available in the art.The ability of such probes and primers to specifically hybridize toEhrlichia chaffeensis polynucleotide sequences will enable them to be ofuse in detecting the presence of complementary sequences in a given testsample. Polynucleotide probes and primers of the invention can hybridizeto complementary sequences in a test sample such as a biological sample,including saliva, sputum, blood, plasma, serum, urine, feces,cerebrospinal fluid, amniotic fluid, wound exudate, or tissue.Polynucleotides from the sample can be, for example, subjected to gelelectrophoresis or other size separation techniques or can beimmobilized without size separation. The polynucleotide probes orprimers can be labeled. Suitable labels, and methods for labeling probesand primers, are known in the art, and include, for example, radioactivelabels incorporated by nick translation or by kinase, biotin labels,fluorescent labels, chemiluminescent labels, bioluminescent labels,metal chelator labels and enzyme labels. The polynucleotides from thesample are contacted with the probes or primers under hybridizationconditions of suitable stringencies.

Depending on the application, varying conditions of hybridization can beused to achieve varying degrees of selectivity of the probe or primertowards the target sequence. For applications requiring highselectivity, relatively stringent conditions can be used, such as lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. For applications requiring lessselectivity, less stringent hybridization conditions can be used. Forexample, salt conditions from about 0.14 M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. The presence of ahybridized complex comprising the probe or primer and a complementarypolynucleotide from the test sample indicates the presence of Ehrlichiachaffeensis polynucleotide in the sample.

Antibodies

Antibodies of the invention are antibody molecules that specificallybind to an Ehrlichia chaffeensis polypeptide of the invention, variantpolypeptides of the invention, or fragments thereof. An antibody of theinvention can be specific for an Ehrlichia chaffeensis polypeptide, forexample, an antibody specific for one or more of SEQ ID NOs:1-5. Inanother embodiment of the invention an antibody is specific for anEhrlichia chaffeensis polypeptide and is not specific for an Ehrlichiacanis polypeptide (e.g., an antibody specific for SEQ ID NOs:1-2 or4-5). In another embodiment of the invention an antibody is specific foran Ehrlichia chaffeensis and an Ehrlichia canis polypeptide (e.g., anantibody specific for SEQ ID NO:3). One of skill in the art can easilydetermine if an antibody is specific for an Ehrlichia chaffeensis or E.canis polypeptide using assays described herein. An antibody of theinvention can be a polyclonal antibody, a monoclonal antibody, a singlechain antibody (scFv), or an antigen binding fragment of an antibody.Antigen-binding fragments of antibodies are a portion of an intactantibody comprising the antigen binding site or variable region of anintact antibody, wherein the portion is free of the constant heavy chaindomains of the Fc region of the intact antibody. Examples of antigenbinding antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂ and F_(v)fragments.

An antibody of the invention can be any antibody class, including forexample, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereofbinds to an epitope of a polypeptide of the invention. An antibody canbe made in vivo in suitable laboratory animals or in vitro usingrecombinant DNA techniques. Means for preparing and characterizingantibodies are well know in the art. See, e.g., Dean, Methods Mol. Biol.80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg,Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol.32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993);Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev.Immunol. 12:125-68 (1992). For example, polyclonal antibodies can beproduced by administering a polypeptide of the invention to an animal,such as a human or other primate, mouse, rat, rabbit, guinea pig, goat,pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animalis collected and the antibodies are purified from the plasma by, forexample, precipitation with ammonium sulfate, followed bychromatography, such as affinity chromatography. Techniques forproducing and processing polyclonal antibodies are known in the art.

“Specifically binds,” “specifically bind” or “specific for” means that afirst antigen, e.g., an Ehrlichia chaffeensis polypeptide, recognizesand binds to an antibody of the invention with greater affinity than toother, non-specific molecules. “Specifically binds.” “specifically bind”or “specific for” also means a first antibody, e.g., an antibody raisedagainst SEQ ID NOs:1-5, recognizes and binds to SEQ ID NOs:1-5, withgreater affinity than to other non-specific molecules. A non-specificmolecule is an antigen that shares no common epitope with the firstantigen. In a preferred embodiment of the invention a non-specificmolecule is not derived from Ehrlichia sp., and in particular is notderived from Ehrlichia chaffeensis or Ehrlichia canis. “Ehrlichia sp.”refers to all species of the genus Ehrlichia. For example, an antibodyraised against a first antigen (e.g., a polypeptide) to which it bindsmore efficiently than to a non-specific antigen can be described asspecifically binding to the first antigen. In one embodiment, anantibody or antigen-binding portion thereof specifically binds to apolypeptide of SEQ ID NOs:1-5 or fragments thereof when it binds with abinding affinity K_(a) of 10⁷ l/mol or more. Specific binding can betested using, for example, an enzyme-linked immunosorbant assay (ELISA),a radioimmunoassay (RIA), or a western blot assay using methodology wellknown in the art.

Antibodies of the invention include antibodies and antigen bindingfragments thereof that (a) compete with a reference antibody for bindingto SEQ ID NOs:1-5 or antigen binding fragments thereof, (b) binds to thesame epitope of SEQ ID NOs:1-5 or antigen binding fragments thereof as areference antibody; (c) binds to SEQ ID NOs:1-5 or antigen bindingfragments thereof with substantially the same K_(d) as a referenceantibody; and/or (d) binds to SEQ ID NOs:1-5 or fragments thereof withsubstantially the same off rate as a reference antibody, wherein thereference antibody is an antibody or antigen-binding fragment thereofthat specifically binds to a polypeptide of SEQ ID NOs:1-5 or antigenbinding fragments thereof with a binding affinity K_(a) of 10⁷ l/mol ormore.

Additionally, monoclonal antibodies directed against epitopes present ona polypeptide of the invention can also be readily produced. Forexample, normal B cells from a mammal, such as a mouse, which wasimmunized with a polypeptide of the invention can be fused with, forexample, HAT-sensitive mouse myeloma cells to produce hybridomas.Hybridomas producing Ehrlichia-specific antibodies can be identifiedusing RIA or ELISA and isolated by cloning in semi-solid agar or bylimiting dilution. Clones producing Ehrlichia-specific antibodies areisolated by another round of screening. Monoclonal antibodies can bescreened for specificity using standard techniques, for example, bybinding a polypeptide of the invention to a microtiter plate andmeasuring binding of the monoclonal antibody by an ELISA assay.Techniques for producing and processing monoclonal antibodies are knownin the art. See e.g., Kohler & Milstein, Nature, 256:495 (1975).Particular isotypes of a monoclonal antibody can be prepared directly,by selecting from the initial fusion, or prepared secondarily, from aparental hybridoma secreting a monoclonal antibody of a differentisotype by using a sib selection technique to isolate class-switchvariants. See Steplewski et al, P.N.A.S. U.S.A. 82:8653 1985; Spria etal., J. Immunolog. Meth. 74:307, 1984. Monoclonal antibodies of theinvention can also be recombinant monoclonal antibodies. See, e.g., U.S.Pat. No. 4,474,893; U.S. Pat. No. 4,816,567. Antibodies of the inventioncan also be chemically constructed. See, e.g., U.S. Pat. No. 4,676,980.

Antibodies of the invention can be chimeric (see, e.g., U.S. Pat. No.5,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986);Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol.2:593 (1992)), caninized, canine, or human antibodies. Human antibodiescan be made by, for example, direct immortilization, phage display,transgenic mice, or a Trimera methodology, see e.g., Reisener et al.,Trends Biotechnol. 16:242-246 (1998).

Antibodies that specifically bind Ehrlichia chaffeensis antigens to theexclusion of E. canis antigens (e.g., SEQ ID NOs:1, 2, 4, and 5) areparticularly useful for detecting the presence of Ehrlichia chaffeensisantigens in a sample, such as a serum, blood, plasma, urine, fecal,cell, tissue, or saliva sample from an animal. Antibodies thatspecifically bind Ehrlichia chaffeensis antigens and E. canis antigens(e.g., SEQ ID NO:3) are particularly useful for detecting the presenceof Ehrlichia chaffeensis and E. canis antigens in a sample. Animmunoassay for can utilize one antibody or several antibodies. Animmunoassay can use, for example, a monoclonal antibody specific for oneepitope, a combination of monoclonal antibodies specific for epitopes ofone polypeptide, monoclonal antibodies specific for epitopes ofdifferent polypeptides, polyclonal antibodies specific for the sameantigen, polyclonal antibodies specific for different antigens, or acombination of monoclonal and polyclonal antibodies. Immunoassayprotocols can be based upon, for example, competition, direct reaction,or sandwich type assays using, for example, labeled antibody. Antibodiesof the invention can be labeled with any type of label known in the art,including, for example, fluorescent, chemiluminescent, radioactive,enzyme, colloidal metal, radioisotope and bioluminescent labels. In oneembodiment of the invention, antibodies of the invention specificallybind Ehrlichia chaffeensis antigens and do not specifically bind toEhrlichia canis antigens (e.g., antibodies specific for SEQ ID NOs:1-2or 4-5). In another embodiment of the invention, antibodies of theinvention specifically bind Ehrlichia chaffeensis antigens andspecifically bind to Ehrlichia canis antigens (e.g., antibodies specificfor SEQ ID NO:3).

Antibodies of the invention or antigen-binding fragments thereof can bebound to a support and used to detect the presence of Ehrlichiachaffeensis and/or E. canis antigens. Supports include, for example,glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagletite.

Antibodies of the invention can further be used to isolate Ehrlichiachaffeensis and/or E. canis organisms or antigens by immunoaffinitycolumns. The antibodies can be affixed to a solid support by, forexample, adsorbtion or by covalent linkage so that the antibodies retaintheir immunoselective activity. Optionally, spacer groups can beincluded so that the antigen binding site of the antibody remainsaccessible. The immobilized antibodies can then be used to bindEhrlichia chaffeensis and/or E. canis organisms or Ehrlichia chaffeensisand/or E. canis antigens from a sample, such as a biological sampleincluding saliva, serum, sputum, blood, urine, feces, cerebrospinalfluid, amniotic fluid, wound exudate, or tissue. The bound Ehrlichiaorganisms or Ehrlichia antigens are recovered from the column matrix by,for example, a change in pH.

Antibodies of the invention can also be used in immunolocalizationstudies to analyze the presence and distribution of a polypeptide of theinvention during various cellular events or physiological conditions.Antibodies can also be used to identify molecules involved in passiveimmunization and to identify molecules involved in the biosynthesis ofnon-protein antigens. Identification of such molecules can be useful invaccine development. Antibodies of the invention, including, forexample, monoclonal antibodies and single chain antibodies, can be usedto monitor the course of amelioration of a disease caused by Ehrlichiachaffeensis. By measuring the increase or decrease of antibodiesspecific for Ehrlichia chaffeensis in a test sample from an animal, itcan be determined whether a particular therapeutic regiment aimed atameliorating the disorder is effective. Antibodies can be detectedand/or quantified using for example, direct binding assays such as RIA,ELISA, or western blot assays.

Methods of Detection

The methods of the invention can be used to detect antibodies orantigen-binding antibody fragments specific for Ehrlichia chaffeensisantigens, Ehrlichia canis antigens, Ehrlichia chaffeensispolynucleotides, E. canis polypeptides or combinations thereof in a testsample, such as a biological sample, an environmental sample, or alaboratory sample. A test sample can potentially comprise Ehrlichia sp.polynucleotides, Ehrlichia chaffeensis polynucleotides, Ehrlichia canispolynucleotides, Ehrlichia sp. polypeptides, Ehrlichia chaffeensispolypeptides, Ehrlichia canis polypeptides, antibodies specific forEhrlichia sp., antibodies specific for Ehrlichia chaffeensis, and/orantibodies specific for Ehrlichia canis, combinations thereof, unrelatedantibodies, polypeptide, polynucleotides, or none of the above. Abiological sample can include, for example, sera, saliva, urine, feces,blood, cells, plasma, or tissue from a mammal such as a horse, cat, dogor human. The test sample can be untreated, precipitated, fractionated,separated, diluted, concentrated, or purified.

In one embodiment methods of the invention comprise contacting one ormore polypeptides of the invention with a test sample under conditionsthat allow polypeptide/antibody complexes, i.e., immunocomplexes, toform. That is, polypeptides of the invention specifically bind toantibodies specific for Ehrlichia chaffeensis and/or E. canis antigenslocated in the sample. In one embodiment of the invention one or morepolypeptides of the invention specifically bind to antibodies that arespecific for Ehrlichia chaffeensis antigens and do not specifically bindto Ehrlichia canis antigens (e.g., SEQ ID NOs:1-2 and 4-5). One of skillin the art is familiar with assays and conditions that are used todetect antibody/polypeptide complex binding. The formation of a complexbetween polypeptides and antibodies in the sample is detected. Theformation of antibody/polypeptide complexes is an indication thatEhrlichia chaffeensis polypeptides and/or Ehrlichia canis polypeptidesare present in the sample. The lack of detection of thepolypeptide/antibody complexes is an indication that Ehrlichiachaffeensis polypeptides and/or Ehrlichia canis polypeptides are notpresent in the sample.

Antibodies of the invention can be used in a method of the diagnosis ofEhrlichia chaffeensis and/or E. canis infection by obtaining a testsample from, e.g., a human or animal suspected of having an Ehrlichiachaffeensis and/or E. canis infection. The test sample is contacted withantibodies of the invention under conditions enabling the formation ofantibody-antigen complexes (i.e., immunocomplexes). One of skill in theart is aware of conditions that enable and are appropriate for formationof antigen/antibody complexes. The amount of antibody-antigen complexescan be determined by methodology known in the art. A level that ishigher than that formed in a negative control sample indicates anEhrlichia chaffeensis and/or E. canis infection. A negative controlsample is a sample that does not comprise any Ehrlichia chaffeensisand/or Ehrlichia canis polypeptides or antibodies specific for Ehrlichiachaffeensis and/or Ehrlichia canis. In one embodiment of the inventionthe negative control contains no Ehrlichia sp. polypeptides orantibodies specific for Ehrlichia sp. In one embodiment of the inventionan antibody is specific for Ehrlichia chaffeensis antigens and is notspecific for Ehrlichia canis antigens. Alternatively, a polypeptide ofthe invention can be contacted with a test sample. Antibodies specificEhrlichia chaffeensis and/or E. canis in a positive test sample willform antigen-antibody complexes under suitable conditions. The amount ofantibody-antigen complexes can be determined by methods known in theart.

In one embodiment of the invention, Ehrlichia chaffeensis and/orEhrlichia canis infection can be detected in a subject. A biologicalsample is obtained from the subject. One or more purified polypeptidescomprising SEQ ID NOs:1-5 or other polypeptides of the invention arecontacted with the biological sample under conditions that allowpolypeptide/antibody complexes to form. The polypeptide/antibodycomplexes are detected. The detection of the polypeptide/antibodycomplexes is an indication that the mammal has an Ehrlichia chaffeensisand/or Ehrlichia canis infection. The lack of detection of thepolypeptide/antibody complexes is an indication that the mammal does nothave an Ehrlichia chaffeensis infection or an Ehrlichia canis infection.

Because SEQ ID NO:3 is specific for both anti-Ehrlichia chaffeensis andanti-Ehrlichia canis antibodies, the detected infection can be Ehrlichiachaffeensis infection, Ehrlichia canis infection, or both Ehrlichiachaffeensis and Ehrlichia canis infection. Because SEQ ID NOs:1, 2, 4,and 5 are specific for anti-Ehrlichia chaffeensis antibodies, thedetected infection is an Ehrlichia chaffeensis infection. The lack ofdetection of polypeptide/antibody complexes is an indication that thesubject does not have an Ehrlichia chaffeensis or an Ehrlichia canisinfection.

In one embodiment of the invention, Ehrlichia chaffeensis and/orEhrlichia canis infection can be detected in a subject by about 5 days,6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days ormore after the subject acquired the Ehrlichia chaffeensis and/orEhrlichia canis infection. In one embodiment of the invention, Ehrlichiachaffeensis and/or Ehrlichia canis infection can be detected in asubject by about 21 days, 20 days, 19 days, 18 days, 17 days, 16 days,15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7days, 6 days, 5 days, or less after the subject acquired the Ehrlichiachaffeensis and/or Ehrlichia canis infection.

In one embodiment of the invention, the polypeptide/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent comprising a signal generating compound can beapplied to the polypeptide/antibody complex under conditions that allowformation of a polypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

In one embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. One or more antibodies thatspecifically bind polypeptides of the invention are added. Theantibodies can be the same antibodies used on the solid phase or can befrom a different source or species and can be linked to an indicatorreagent, such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

In another embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. Second anti-species antibodies thatspecifically bind polypeptides of the invention are added. These secondantibodies are from a different species than the solid phase antibodies.Third anti-species antibodies are added that specifically bind thesecond antibodies and that do not specifically bind the solid phaseantibodies are added. The third antibodies can comprise and indicatorreagent such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

Assays of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to enzyme linked immunosorbent assay (ELISA), western blot, IFA,radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), and microtiter plate assays (any assay done in oneor more wells of a microtiter plate). One assay of the inventioncomprises a reversible flow chromatographic binding assay, for example aSNAP® assay. See e.g., U.S. Pat. No. 5,726,010.

Assays can use solid phases or substrates or can be performed byimmunoprecipitation or any other methods that do not utilize solidphases. Where a solid phase or substrate is used, one or morepolypeptides of the invention are directly or indirectly attached to asolid support or a substrate such as a microtiter well, magnetic bead,non-magnetic bead, column, matrix, membrane, fibrous mat composed ofsynthetic or natural fibers (e.g., glass or cellulose-based materials orthermoplastic polymers, such as, polyethylene, polypropylene, orpolyester), sintered structure composed of particulate materials (e.g.,glass or various thermoplastic polymers), or cast membrane film composedof nitrocellulose, nylon, polysulfone or the like (generally syntheticin nature). In one embodiment of the invention a substrate is sintered,fine particles of polyethylene, commonly known as porous polyethylene,for example, 10-15 micron porous polyethylene from Chromex Corporation(Albuquerque, N. Mex.). All of these substrate materials can be used insuitable shapes, such as films, sheets, or plates, or they may be coatedonto or bonded or laminated to appropriate inert carriers, such aspaper, glass, plastic films, or fabrics. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like.

In one type of assay format, one or more polypeptides can be coated on asolid phase or substrate. A test sample suspected of containinganti-Ehrlichia chaffeensis antibodies and/or E. canis antibodies orantigen-binding fragments thereof is incubated with an indicator reagentcomprising a signal generating compound conjugated to an antibodies orantibody fragments specific for Ehrlichia chaffeensis and/or E. canisfor a time and under conditions sufficient to form antigen/antibodycomplexes of either antibodies of the test sample to the polypeptides ofthe solid phase or the indicator reagent compound conjugated to anantibody specific for Ehrlichia chaffeensis and/or E. canis to thepolypeptides of the solid phase. The reduction in binding of theindicator reagent conjugated to anti-Ehrlichia chaffeensis and/or E.canis antibodies to the solid phase can be quantitatively measured. Ameasurable reduction in the signal compared to the signal generatedfrom, e.g., a confirmed negative Ehrlichia chaffeensis test sampleindicates the presence of anti-Ehrlichia chaffeensis antibodies in thetest sample. This type of assay can quantitate the amount ofanti-Ehrlichia chaffeensis and/or E. canis antibodies in a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. Ifantibodies specific for Ehrlichia chaffeensis and/or E. canis arepresent in the test sample they will bind the one or more polypeptidesconjugated to an indicator reagent and to the one or more polypeptidesimmobilized on the support. The polypeptide/antibody/indicator complexcan then be detected. This type of assay can quantitate the amount ofanti-Ehrlichia chaffeensis and/or E. canis antibodies in a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. The test sample isapplied to the support or substrate and incubated. Unbound componentsfrom the sample are washed away by washing the solid support with a washsolution. If Ehrlichia chaffeensis specific antibodies are present inthe test sample, they will bind to the polypeptide coated on the solidphase. This polypeptide/antibody complex can be detected using a secondspecies-specific antibody that is conjugated to an indicator reagent.The polypeptide/antibody/anti-species antibody indicator complex canthen be detected. This type of assay can quantitate the amount ofanti-Ehrlichia chaffeensis antibodies in a test sample.

The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by, e.g.,radiometric, calorimetric, fluorometric, size-separation, orprecipitation methods. Optionally, detection of a polypeptide/antibodycomplex is by the addition of a secondary antibody that is coupled to anindicator reagent comprising a signal generating compound. Indicatorreagents comprising signal generating compounds (labels) associated witha polypeptide/antibody complex can be detected using the methodsdescribed above and include chromogenic agents, catalysts such as enzymeconjugates fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Formation of the complex is indicative of the presence of anti-Ehrlichiachaffeensis and/or E. canis antibodies in a test sample. Therefore, themethods of the invention can be used to diagnose Ehrlichia chaffeensisand/or E. canis infection in an animal.

The methods of the invention can also indicate the amount or quantity ofanti-anti-Ehrlichia chaffeensis and/or E. canis antibodies in a testsample. With many indicator reagents, such as enzyme conjugates, theamount of antibody present is proportional to the signal generated.Depending upon the type of test sample, it can be diluted with asuitable buffer reagent, concentrated, or contacted with a solid phasewithout any manipulation. For example, it usually is preferred to testserum or plasma samples that previously have been diluted, orconcentrated specimens such as urine, in order to determine the presenceand/or amount of antibody present.

The invention further comprises assay kits (e.g., articles ofmanufacture) for detecting anti-Ehrlichia chaffeensis and/or E. canisantibodies or antigen-binding antibody fragments, or Ehrlichiachaffeensis and/or E. canis polypeptides in a sample. A kit comprisesone or more polypeptides of the invention and means for determiningbinding of the polypeptide to anti-Ehrlichia chaffeensis and/or E. canisantibodies or antibody fragments in the sample. A kit or article ofmanufacture can also comprise one or more antibodies or antibodyfragments of the invention and means for determining binding of theantibodies or antibody fragments to Ehrlichia chaffeensis and/or E.canis polypeptides in the sample. A kit can comprise a device containingone or more polypeptides or antibodies of the invention and instructionsfor use of the one or more polypeptides or antibodies for, e.g., theidentification of an Ehrlichia chaffeensis and/or E. canis infection ina mammal. The kit can also comprise packaging material comprising alabel that indicates that the one or more polypeptides or antibodies ofthe kit can be used for the identification of Ehrlichia chaffeensisand/or E. canis infection. Other components such as buffers, controls,and the like, known to those of ordinary skill in art, can be includedin such test kits. The polypeptides, antibodies, assays, and kits of theinvention are useful, for example, in the diagnosis of individual casesof Ehrlichia chaffeensis and/or E. canis infection in a patient, as wellas epidemiological studies of Ehrlichia chaffeensis and/or E. canisoutbreaks.

Polypeptides and assays of the invention can be combined with otherpolypeptides or assays to detect the presence of Ehrlichia chaffeensisand/or E. canis along with other organisms. For example, polypeptidesand assays of the invention can be combined with reagents that detectheartworm and/or Borrelia burgdorferi and/or Anaplasma platys and/orAnaplasma phagocytophilum.

Polynucleotides of the invention can be used to detect the presence ofEhrlichia chaffeensis polynucleotides in a sample. The polynucleotidescan be used to detect Ehrlichia chaffeensis polynucleotides in a sampleby a simple hybridization reaction and can also be used in, e.g.,polymerase chain reactions (PCR) such as a real-time PCR reaction.Methods and compositions of the invention can also be used todifferentially detect the presence Ehrlichia chaffeensis from otherEhrlichia sp., such as Ehrlichia canis.

PCR assays are well described in the art, including, for example, U.S.Pat. No. 4,683,195; U.S. Pat. No. 4,683,202;U.S. Pat. No. 4,965,188.Generally, polynucleotide primers are annealed to denatured strands of atarget nucleic acid. Primer extension products are formed bypolymerization of deoxynucleoside triphosphates by a polymerase. PCRthen involves repetitive cycles of template nucleic acid denaturation,primer annealing and extension of the annealed primers by the action ofa thermostable polymerase. The process results in exponentialamplification of the target Ehrlichia chaffeensis and/or E. canisnucleic acids in the test sample, which allows for the detection oftarget polynucleotides existing in very low concentrations in a sample.

Real-time PCR assays are based on the detection of a signal, e.g., afluorescent reporter signal. This signal increases in direct proportionto the amount of PCR product in a reaction. Real-time PCR is anyamplification technique that makes it possible to monitor the evolutionof an ongoing amplification reaction. See, Quantitation of DNA/RNA UsingReal-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCRProtocols (Academic Press New York, 1989). By recording the amount offluorescence emission at each cycle, it is possible to monitor the PCRreaction during exponential phase where the first significant increasein the amount of PCR product correlates to the initial amount of targettemplate. The higher the starting copy number of the nucleic acidtarget, the sooner a significant increase in fluorescence is observed.

One embodiment of the invention provides a method for detecting and/orquantifying Ehrlichia chaffeensis and/or E. canis polynucleotides in atest sample. Sense primers and antisense primers can be added to a testsample under conditions suitable for a polymerase chain reaction. Theprimers hybridize with Ehrlichia chaffeensis and/or E. canispolynucleotides such that an amplification product is formed ifEhrlichia chaffeensis and/or E. canis polynucleotides are present in thetest sample. Amplification products are detected and the presence and/orquantity of Ehrlichia chaffeensis and/or E. canis polynucleotides isdetermined. Amplification products can be detected with a polynucleotideprobe that hybridizes, under conditions suitable for a polymerase chainreaction, with an Ehrlichia chaffeensis and/or E. canis polynucleotidesequence. The amplification product can be quantified by measuring adetection signal from the probe and comparing said detection signal to asecond probe detection signal from a quantification standard. Thequantification standard can be extracted in parallel with the testsample.

Methods of Treatment, Amelioration, or Prevention of a Disease Caused byE. chaffeensis or E. canis

Polypeptides, polynucleotides, and antibodies of the invention can beused to treat, ameliorate, or prevent a disease caused by E. chaffeensisand/or E. canis.

For example, an antibody, such as a monoclonal antibody of the inventionor antigen-binding fragments thereof, can be administered to an animal,such as a human or dog. In one embodiment of the invention an antibodyor antigen-binding fragment thereof is administered to an animal in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. A pharmaceutical composition comprises a therapeuticallyeffective amount of an antibody or antigen-binding fragments thereof. Atherapeutically effective amount is an amount effective in alleviatingthe symptoms of an E. chaffeensis and/or E. canis infection or inreducing the amount of E. chaffeensis and/or E. canis organisms in asubject.

Polypeptides or polynucleotides of the invention can be present in animmunogenic composition and used to elicit an immune response in a host.An immunogenic composition or immunogen is capable of inducing an immuneresponse in an animal. An immunogenic polypeptide or polynucleotidecomposition of the invention is particularly useful in sensitizing animmune system of an animal such that, as one result, an immune responseis produced that ameliorates or prevents the effect of E. chaffeensisand/or E. canis infection. The elicitation of an immune response inanimal model can be useful to determine, for example, optimal doses oradministration routes. Elicitation of an immune response can also beused to treat, prevent, or ameliorate a disease or infection caused byE. chaffeensis and/or E. canis. An immune response includes humoralimmune responses or cell mediated immune responses, or a combinationthereof. An immune response can also comprise the promotion of ageneralized host response, e.g., by promoting the production ofdefensins.

One embodiment of the invention provides an immunogen that comprises apolypeptide of the invention and one or more additional regions ormoieties covalently joined to the polypeptide at the carboxyl terminusor amino terminus. Each region or moiety can, for example, enhance theimmune response, facilitate purification of the immunogen, or facilitatepolypeptide stability.

The generation of an antibody titer by an animal against E. chaffeensisand/or E. canis can be important in protection from infection andclearance of infection. Detection and/or quantification of antibodytiters after delivery of a polypeptide or polynucleotide can be used toidentify epitopes that are particularly effective at eliciting antibodytiters. Epitopes responsible for a strong antibody response to E.chaffeensis and/or E. canis can be identified by eliciting antibodiesdirected against E. chaffeensis and/or E. canis polypeptides ofdifferent lengths. Antibodies elicited by a particular polypeptideepitope can then be tested using, for example, an ELISA assay todetermine which polypeptides contain epitopes that are most effective atgenerating a strong response. Polypeptides or fusion proteins thatcontain these epitopes or polynucleotides encoding the epitopes can thenbe constructed and used to elicit a strong antibody response.

A polypeptide, polynucleotide, or antibody of the invention can beadministered to a mammal, such as a mouse, rabbit, guinea pig, macaque,baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or toanimals such as chickens or ducks, to elicit antibodies in vivo.Injection of a polynucleotide has the practical advantages of simplicityof construction and modification. Further, injection of a polynucleotideresults in the synthesis of a polypeptide in the host. Thus, thepolypeptide is presented to the host immune system with nativepost-translational modifications, structure, and conformation. Apolynucleotide can be delivered to a subject as “naked DNA.”

Administration of a polynucleotide, polypeptide, or antibody can be byany means known in the art, including intramuscular, intravenous,intrapulmonary, intramuscular, intradermal, intraperitoneal, orsubcutaneous injection, aerosol, intranasal, infusion pump, suppository,mucosal, topical, and oral, including injection using a biologicalballistic gun (“gene gun”). A polynucleotide, polypeptide, or antibodycan be accompanied by a protein carrier for oral administration. Acombination of administration methods can also be used to elicit animmune response. Antibodies can be administered at a daily dose of about0.5 mg to about 200 mg. In one embodiment of the invention antibodiesare administered at a daily dose of about 20 to about 100 mg.

Pharmaceutically acceptable carriers and diluents and veterinarilyacceptable carries and diluents for therapeutic use are well known inthe art and are described in, for example, Remington's PharmaceuticalSciences, Mack Publishing Co. (A. R. Gennaro ed. (1985)). The carriershould not itself induce the production of antibodies harmful to thehost. Such carriers include, but are not limited to, large, slowlymetabolized, macromolecules, such as proteins, polysaccharides such aslatex functionalized SEPHAROSE®, agarose, cellulose, cellulose beads andthe like, polylactic acids, polyglycolic acids, polymeric amino acidssuch as polyglutamic acid, polylysine, and the like, amino acidcopolymers, peptoids, lipitoids, and inactive, avirulent virus particlesor bacterial cells. Liposomes, hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesives can also be used as a carrier for acomposition of the invention.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, phosphate buffered saline, Ringer's solution,Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, orthe like, singly or in combination, as well as substances such aswetting agents, emulsifying agents, tonicity adjusting agents,detergent, or pH buffering agents. Additional active agents, such asbacteriocidal agents can also be used.

If desired, co-stimulatory molecules, which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asMIP1α, GM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants are substances that can be used to nonspecifically augment aspecific immune response. Generally, an adjuvant and a polypeptide ofthe invention are mixed prior to presentation to the immune system, orpresented separately, but are presented into the same site of theanimal. Adjuvants can include, for example, oil adjuvants (e.g. Freund'scomplete and incomplete adjuvants) mineral salts (e.g. Alk(SO₄)₂;AlNa(SO₄)₂, AlNH₄(SO₄), Silica, Alum, Al(OH)₃, and Ca₃(PO₄)₂),polynucleotides (i.e. Poly IC and Poly AU acids), and certain naturalsubstances (e.g. wax D from Mycobacterium tuberculosis, as well assubstances found in Corynebacterium parvum, Bordetella pertussis andmembers of the genus Brucella. Adjuvants which can be used include, butare not limited to MF59-0, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 (polysorbate) emulsion.

The compositions of the invention can be formulated into ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, injectable formulations, mouthwashes, dentrifices, andthe like. The percentage of one or more polypeptides, polynucleotides,or antibodies of the invention in such compositions and preparations canvary from 0.1% to 60% of the weight of the unit.

Administration of polypeptides, polynucleotides, or antibodies canelicit an immune response in the animal that lasts for at least 1 week,1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immuneresponse can be maintained in an animal by providing one or more boosterinjections of the polypeptide, polynucleotide, or antibodies at 1 month,3 months, 6 months, 1 year, or more after the primary injection. Ifdesired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

A composition of the invention comprising a polypeptide, polynucleotide,antibody, or a combination thereof is administered in a mannercompatible with the particular composition used and in an amount that iseffective to elicit an immune response as detected by, for example, anELISA. A polynucleotide can be injected intramuscularly to a mammal,such as a baboon, chimpanzee, dog, or human, at a dose of 1 ng/kg, 10ng/kg, 100 ng/kg, 1000 ng/kg, 0.001 mg/kg, 0.1 mg/kg, or 0.5 mg/kg. Apolypeptide or antibody can be injected intramuscularly to a mammal at adose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

Polypeptides, polynucleotides, or antibodies, or a combination thereofcan be administered either to an animal that is not infected with E.chaffeensis and/or E. canis or can be administered to an E. chaffeensisand/or E. canis-infected animal. An immunologically effective amount ortherapeutically effective amount means the administration of that amountto an individual, either in a single dose or as part of series, iseffective for treatment, amelioration, or prevention of E. chaffeensisand/or E. canis infection. The particular dosages of polynucleotide,polypeptides, or antibodies in a composition will depend on many factorsincluding, but not limited to the species, age, gender, concurrentmedication, general condition of the mammal to which the composition isadministered, and the mode of administration of the composition. Aneffective amount of the composition of the invention can be readilydetermined using only routine experimentation.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms, while retaining their ordinary meanings.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by embodiments,optional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

EXAMPLES Example 1 Direct ELISA Assay Plates and Protocols

Polypeptides shown in SEQ ID NO:4 and SEQ ID NO:5 were conjugated to BSAand the conjugate was coated onto IMMULON® 4 plates. The coating bufferwas 0.05M sodium carbonate, pH 9.6. 100 uL/well of diluted polypeptidewas pipetted onto the plates. The plates were covered and incubatedovernight at 2° C.-8° C. The polypeptide solution was aspirated from theplates and the plates were washed 4× with HW PetChek® wash buffer (IDEXXLaboratories, Inc., Westbrook Me.). 300 uL/well of 1% BSA in 0.1M TrispH 7.6 was added to the plates. The plates were incubated, covered, for6 hours at RT. The BSA was aspirated and 300 uL/well of 2.5% sucrose in0.1M Tris pH 7.6 was added to the plates. The plates were incubated,covered, overnight at 2° C.-8° C. The sucrose was aspirated from theplates and the plates were tapped to remove excess liquid. The plateswere dried in a vacuum chamber for 4 hours. The plates were store withdesiccants in double plastic bags at 2° C.-8° C.

Polypeptides shown in SEQ ID NO:4 and SEQ ID NO:5 were conjugated toHRPO. Diluted polypeptide:HRPO was added to each well (100 uL/well) andcontrols and sample (neat) were added (50 uL/well). A positive controlfor E. chaffeensis was used along with a negative control. The plateswere tapped gently and incubated for 1 hour at RT. The plates werewashed 6× with HW PetChek® wash buffer. 100 uL/well of TMB substrate wasadded to the wells and the plates were incubated for 10 min. 50 uL/wellstop solution was added to the wells. The plates were read at A650. Thenegative cutoff was determined as 2× negative control O.D. value.

Example 2 Indirect ELISA Assay Plates and Protocols

Polypeptides shown in SEQ ID NOs:5, 4 and 3 were coated on Immulon® 1plates. The coating buffer was 0.05M sodium carbonate, pH 9.6. 100uL/well diluted peptide was added to the plates and the plates wereincubated, covered, overnight at room temperature (RT). The polypeptideswere aspirated and the plates were washed 2× with HW PetChek® washbuffer. 200 uL/well 2% TWEEN® (polysorbate) 20/2.5% sucrose in 0.1M TrispH 7.6 was added to the wells and the plates were incubated, covered,for 2 hours at RT. The blocking solution was aspirated and the plateswere tapped to remove excess liquid. The plates were dried in mylar bagsovernight at RT with 2 (27 g) desiccants/6 plates. The plates werestored at 2° C.-8° C.

Diluted controls and samples were pipetted into the wells at 100uL/well. A positive control for E. chaffeensis was used along with anegative control. The plates were incubated for 30 min. at RT. Theplates were washed 5× with HW PetChek® wash buffer. 100 uL/well ofdiluted Rabbit anti-dog HRPO was added to the wells and incubated for 30min. at RT. The plates were washed 5× with HW PetChek® wash buffer. 50uL/well of TMB substrate was added to the plates and they were incubatedfor 10 min. 50 uL/well of stop solution was added. The plates were readat A650. The negative cutoff was determined as 2× negative control O.D.value.

Example 3

p120 Polypeptide Assays

p120B (SEQ ID NO:4) was used in direct assays as described above toassay E. canis vaccinated dog samples (the dogs were vaccinated asdescribed in US Patent Publication No. 20060234322). These assays weredone to determine if p120B (SEQ ID NO:4) would provide a positive resultin E. canis vaccinated dogs. The test samples were taken from the dogsafter the second booster vaccination. The plates were coated at 0.5ug/mL and the peptide:HRPO was used at a concentration of 1 ug/mL. TheSNAP® 4Dx® assay was used to show that an anti-E. canis antibodyresponse was induced in the vaccinated dogs. This assay screens forheartworm antigen, Ehrlichia canis antibody, Borrelia burgdorferiantibody, and Anaplasma phagocytophilum antibody. The results are shownin Table 1. Positive results for E. canis antibody in the SNAP® 4Dx®test indicate that an antibody response was induced followingvaccination. p120B (SEQ ID NO:4) does not provide a positive result whentested with samples from E. canis vaccinated dogs in direct assays.Therefore, p120B (SEQ ID NO:4) is specific for E. chaffeensis infectionand does not react with sera from E. canis vaccinated dogs.

TABLE 1 SNAP ® 4Dx ® for E. canis Ab Plate assay Signal result minusP120B Sample Bckgrnd (SEQ ID NO: 4) CVYDEH Day 70 N 0.039 Ribi Day 1050.07 0.035 (vw+) Day 112 0.17 0.035 Day 126 0.18 0.035 CWMBDC Day 700.08 0.036 Ribi Day 105 0.45 0.037 Day 112 0.40 0.034 Day 126 0.30 0.034CVXCSM Day 70 N 0.033 Ribi Day 105 N 0.037 Day 112 0.14 0.035 Day 1260.23 0.035 CWMAXK Day 70 0.07 0.038 Ribi + BCG (vw+) Day 105 0.26 0.035Day 112 0.36 0.035 Day 126 0.34 0.034 CVSCVA Day 70 0.10 (w+) 0.037Ribi + BCG Day 105 0.51 0.035 Day 112 0.45 0.034 Day 126 0.47 0.035CVXCAP Day 70 N 0.034 Ribi + BCG Day 105 0.51 0.034 Day 112 0.42 0.037Day 126 0.48 0.034

p120B (SEQ ID NO:4) and p120-R (SEQ ID NO:3) were used in an indirectassay as described above to test samples from dogs experimentallyinfected with E. canis. This assay was done to determine if p120B (SEQID NO:4) and p120-R (SEQ ID NO:3) would provide a positive result insamples from E. canis infected dogs. The plates were coated at 0.5ug/ml. The sample dilution was 1:50 for p120B (SEQ ID NO:4) and 1:100for p120-R (SEQ ID NO:3). The rabbit anti-dog:HRPO conjugate was used ata 1:1000 dilution for p120B (SEQ ID NO:4) and at a 1:2000 dilution forp120-R (SEQ ID NO:3). The results are shown in Table 2.

TABLE 2 IDX P120B P120-R (SEQ ID NO: 4) (SEQ ID NO: 3) Sample A650Result A650 Result (PC) 21349M 2.342 + 2.074 + (NC) 21172M 0.031 N 0.039N ~Cutoff 0.062 0.078 AVS Pre-bleed 0.029 N 0.036 N Wk 1 0.031 N 0.034 NWk 2 0.028 N 0.164 w+ Wk 3 0.027 N 0.566 + Wk 4 0.028 N 0.721 + Wk 80.030 N 1.161 + ARR Pre-bleed 0.025 N 0.036 N Wk 1 0.029 N 0.040 N Wk 20.023 N 0.107 vw+ Wk 3 0.032 N 0.580 + Wk 4 0.033 N 1.397 + Wk 8 0.034 N1.649 + ASR Pre-bleed 0.028 N 0.025 N Wk 1 0.025 N 0.028 N Wk 2 0.027 N0.441 + Wk 3 0.036 N 1.407 + Wk 4 0.031 N 1.658 + Wk 8 0.032 N 1.833 +ATL Pre-bleed 0.024 N 0.032 N Wk 1 0.032 N 0.037 N Wk 2 0.033 N 0.127 w+Wk 3 0.040 N 0.777 + Wk 4 0.032 N 0.738 + Wk 8 0.038 N 0.577 + AUEPre-bleed 0.028 N 0.035 N Wk 1 0.033 N 0.047 N Wk 2 0.044 N 0.166 w+ Wk3 0.045 N 0.746 + Wk 4 0.033 N 0.754 + Wk 8 0.041 N 0.908 +

p120B (SEQ ID NO:4) does not provide a positive result when tested withsamples from E. canis infected dogs. Therefore, p120B (SEQ ID NO:4) isspecific for E. chaffeensis and not for E. canis. p120-R (SEQ ID NO:3),however, appears to cross react with E. canis. Therefore, p120-R (SEQ IDNO:3) is specific for both E. chaffeensis and E. canis.

p120B (SEQ ID NO:4) and p120-R (SEQ ID NO:3) were used in an indirectassays as described above to test samples from dogs experimentallyinfected with E. chaffeensis at several time points after infection. Theplates were coated at 0.5 ug/ml. The sample dilution was 1:50 for p120B(SEQ ID NO:4) and 1:100 for p120-R (SEQ ID NO:3). The rabbitanti-dog:HRPO conjugate was used at a 1:1000 dilution for p120B (SEQ IDNO:4) and 1:2000 for p120-R (SEQ ID NO:3). The results are shown inTable 3 and FIGS. 1A and 1B. p120B (SEQ ID NO:4) and p120-R (SEQ IDNO:3) were able to detect E. chaffeensis antibodies at least by day 7post-infection.

TABLE 3 p120B p120-R (SEQ ID NO: 4) (SEQ ID NO: 3) Sample A650 ResultA650 Result (PC) 21349M 2.457 + 1.516 + (NC) 21172M 0.045 N 0.047 N~Cutoff 0.090 0.094 CTUALJ Pre-bleed 0.067 N 0.047 N Day 7 0.316 +0.608 + Day 14 0.451 + 1.295 + Day 21 0.344 + 1.016 + Day 28 0.277 +0.928 + Day 36 0.233 + 0.646 + Day 42 0.228 + 0.593 + Day 49 0.217 +0.514 + Day 56 0.198 + 0.507 + Day 63 0.193 + 0.558 + Day 77 0.270 +0.273 + Day 96 0.290 + 0.273 + CURALN Pre-bleed 0.036 N 0.046 N Day 70.666 + 0.785 + Day 14 0.882 + 1.212 + Day 21 0.634 + 0.797 + Day 280.485 + 0.730 + Day 35 0.362 + 0.486 + Day 42 0.299 + 0.427 + Day 490.287 + 0.305 + Day 63 0.294 + 0.260 + Day 82 0.667 + 0.381 + (n = 10)Mean Neg 0.039 N 0.047 N

Synthetic polypeptides p120B (SEQ ID NO:4) and p120BK (SEQ ID NO:5) wereused in direct and indirect assays as described above to test a samplefrom a dog known to be E. chaffeensis antibody positive.

For the direct assay, the peptide-BSA conjugates were coated onto platesat 0.5 ug/ml. The peptide:HRPO conjugates were used at 1 ug/ml. Theresults are shown in Table 4. Both p120B and p120BK showed positive testresults with the sample from the E. chaffeensis-positive dog (ID21349M), but not with the samples from E. chaffeensis-negative control(NC) canines. Therefore, both p120B and p120BK detected E. chaffeensisantibodies in the E. chaffeensis-positive dog.

For the indirect assay, the peptides were coated onto plates at 0.5ug/ml. The sample dilution was 1:100. The rabbit anti-dog:HRPO conjugatewas used at 1:2000 dilution. The results are shown in Table 5. Bothp120B and p120BK showed positive test results with the sample from theE. chaffeensis-positive dog (ID 21349M), but not with the samples fromE. chaffeensis-negative control (NC) canines. Therefore, both p1120B andp120BK were able to detect E. chaffeensis antibodies in the E.chaffeensis-positive dog.

TABLE 4 Sample Plate Results (A650) ID p120 p120BK E. chaffeensis 21349M4.000 2.803 positive NC 21172M 0.035 0.037 NC 21056F 0.035 0.035 NC21067M 0.034 0.036 NC 21069F 0.038 0.038

TABLE 5 Sample Plate Results (A650) ID p120 p120BK E. chaffeensis 21349M1.811 1.337 positive NC 21172M 0.033 0.036 NC 21056F 0.035 0.035 NC21067M 0.037 0.041

1. A purified polypeptide comprising: (a) SEQ ID NO:1, wherein thepolypeptide consists of less than about 50 contiguous naturallyoccurring Ehrlichia chaffeensis amino acids; (b) SEQ ID NO:2, whereinthe polypeptide consists of less than about 50 contiguous naturallyoccurring Ehrlichia chaffeensis amino acids; (c) SEQ ID NO:4, whereinthe polypeptide consists of less than about 50 contiguous naturallyoccurring Ehrlichia chaffeensis amino acids; or (d) SEQ ID NO:5, whereinthe polypeptide consists of less than about 50 contiguous naturallyoccurring Ehrlichia chaffeensis amino acids.
 2. An isolatedpolynucleotide that encodes the purified polypeptide of claim
 1. 3. Thepurified polypeptide of claim 1, wherein the polypeptide consists of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
 4. The purifiedpolypeptide of claim 1, wherein the purified polypeptide is linked to anindicator reagent, an amino acid spacer, a signal sequence, a stoptransfer sequence, a transmembrane domain, a protein purificationligand, a heterologous polypeptide, one or more additional polypeptidescomprising SEQ ID NOs:1, 2, 3, 4, 5, or a combination thereof.
 5. Thepurified polypeptide of claim 1, wherein the purified polypeptidecomprises one or more C amino acid residues at the amino terminus orcarboxy terminus or both termini of the polypeptide.
 6. A method ofdetecting antibodies that specifically bind an Ehrlichia chaffeensispolypeptide in a test sample, comprising: (a) contacting a purifiedpolypeptide comprising SEQ ID NO:1, 2, 3, 4, or 5 with the test sample,under conditions that allow polypeptide/antibody complexes to form;wherein when the purified polypeptide comprises SEQ ID NO:1, 2, 4 or 5,the purified polypeptide consists of less than about 50 contiguousnaturally occurring Ehrlichia chaffeensis amino acids and wherein whenthe purified polypeptide comprises SEQ ID NO:3, the purified polypeptideconsists of less than about 575 contiguous naturally occurring Ehrlichiachaffeensis amino acids; (b) detecting the polypeptide/antibodycomplexes; wherein the detection of the polypeptide/antibody complexesis an indication that antibodies specific for Ehrlichia chaffeensis arepresent in the test sample, and wherein the absence of thepolypeptide/antibody complexes is an indication that antibodies specificfor Ehrlichia chaffeensis are not present in the test sample.
 7. Themethod of claim 6, further comprising contacting the complexes of step(a) with an indicator reagent prior to the performance of step (b). 8.The method of claim 6, wherein the purified polypeptide comprises SEQ IDNOs:1 2, 4, or 5 and wherein the method does not detect antibodies thatspecifically bind an Ehrlichia canis polypeptide.
 9. The method of claim6, wherein the amount of antibody in the test sample is determined. 10.The method of claim 6, wherein the purified polypeptide is attached to asubstrate.
 11. The method of claim 6, wherein the purified polypeptideis linked to an indicator reagent, an amino acid spacer, a signalsequence, a stop transfer sequence, a transmembrane domain, a proteinpurification ligand, a heterologous protein, one or more additionalpolypeptides comprising SEQ ID NOs:1, 2, 3, 4, 5 or a combinationthereof.
 12. A method of detecting an Ehrlichia chaffeensis infection ina subject comprising: (a) obtaining a biological sample from thesubject; (b) contacting a purified polypeptide comprising SEQ ID NO:1,2, 3, 4 or 5 with the biological sample under conditions that allowpolypeptide/antibody complexes to form; wherein when the purifiedpolypeptide comprises SEQ ID NO:1, 2, 4 or 5, the purified polypeptideconsists of less than about 50 contiguous naturally occurring Ehrlichiachaffeensis amino acids, and wherein when the purified polypeptidecomprises SEQ ID NO:3, the purified polypeptide consists of less thanabout 575 contiguous naturally occurring Ehrlichia chaffeensis aminoacids; (c) detecting the polypeptide/antibody complexes; wherein thedetection of the polypeptide/antibody complexes is an indication thatthe subject has an Ehrlichia chaffeensis infection.
 13. The method ofclaim 12, wherein the purified polypeptide is SEQ ID NO:1, 2, 4, or 5and wherein the method does not detect Ehrlichia canis infection in thesubject.
 14. An antibody that specifically binds to a polypeptideconsisting of SEQ ID NO:1, 2, 3, 4 or
 5. 15. The antibody of claim 14,wherein the antibody is a monoclonal antibody, polyclonal antibody,antigen-binding antibody fragment, or a single chain antibody.
 16. Amethod of detecting an Ehrlichia chaffeensis polypeptide in a samplecomprising: (a) contacting antibodies that specifically bind to apolypeptide consisting of SEQ ID NO:1 2, 4, or 5 with the sample underconditions that allow polypeptide/antibody complexes to form; (b)detecting the polypeptide/antibody complexes; wherein the detection ofthe polypeptide/antibody complexes is an indication that an Ehrlichiachaffeensis polypeptide is present in the sample.
 17. The method ofclaim 16, wherein the antibodies are monoclonal antibodies, polyclonalantibodies, antigen-binding antibody fragments, or single chainantibodies.
 18. The method of claim 16, wherein the antibodies areattached to a substrate.